The overall objective of this project is to define the cellular and molecular mechanisms of CD4 T-cell tolerance to a systemic self-antigen. The studies will test the hypotheses that cell-intrinsic tolerance in T-cells results from a combination of proximal signaling blocks and imbalanced cytokine production, and when the cell-intrinsic mechanisms of anergy and deletion fail, prolonged self-antigen recognition without other stimuli may lead to progressive development of regulatory T-cells. The studies will rely on a well-established transgenic model of systemic T-cell tolerance which has some unique strengths, notably that it is amenable to biochemical and molecular analyses of cells that have encountered self-antigen, and it is the only system in which effector and regulatory T-cells are generated sequentially from a monoclonal T-cell population in response to self-antigen recognition in peripheral tissues. The following specific aims will be addressed: 1. Mechanisms of cell-intrinsic tolerance (anergy and deletion) induced by a systemic self-antigen. These studies will define signaling blocks in T-cells rendered anergic by recognition of systemic self-antigen in vivo, using a novel multiplex phosphoprotein array and other techniques, and examine the roles of known T-cell regulators (CTLA-4, Fas, Bim) in systemic tolerance. In addition, the studies will explore the novel idea that imbalanced production of IFN-y without IL-2 contributes to T-cell tolerance, and define the mechanisms underlying this unexpected role of IFN-y as a tolerance-inducing cytokine. 2. Induction and functions of peripherally generated regulatory T-cells (Treg). Using a model of sequential development of effector T-cells and Treg in response to recognition of systemic antigen, these studies will define the lineage relationships between these two cell populations and the roles of cytokines in controlling the balance between effector and regulatory cells. A model of parent to F1 graft-vs-host reaction will be used to examine the development of effector and regulatory T-cells in situations of polyclonal T-cell reactivity. Thus, this project uses a defined experimental system and a variety of precise analytical methods to study fundamental mechanisms of peripheral T-cell tolerance, its induction and maintenance, and its regulation by external signals. There are numerous close interactions between this project and studies of CTLA-4 and Treg, signaling pathways, interactions of central and peripheral tolerance mechanisms. The results are not only of biological significance, but will also provide valuable leads for strategies to induce tolerance as a therapeutic modality.